This invention relates to new and improved apparatus and method for the measurement of the sperm density in a semen specimen and the determination of dosages for artificial insemination.
When properly practiced, artificial insemination (AI) can be a highly successful management technique for improving the reproductive efficiency of various domestic animals.
AI is widely practiced in the cattle breeding industry and is finding increasing use in equine (all breeds except thoroughbred) reproduction; and is being used, at least experimentally in the breeding of canines and other animals.
There are many specific reasons for the practice of AI, most of which ultimately reduce to a matter of economics. For example, an AI program conducted by competent practitioners using proven techniques and equipment can reduce the occurrence of injury to valuable animals and diminish the transmission of certain reproductive system diseases. Additionally, and of major importance, a maximum number of females can be brought into pregnancy in minimum time and the number of progeny from males of recognized genetic superiority can be greatly increased.
Although the detailed procedures and the apparatus used in an AI program vary with the characteristics and anatomy of the various species, the handling, processing and evaluation of semen in the field laboratory is nearly the same for all animals.
In some species, such as the equine, the ejaculate contains a gel-like substance which has no bearing on fertilization but does interfere with laboratory evaluation and insemination procedures. The gel is removed at the time of collection by passing the ejaculate through an appropriate filter prior to its entry into the holding container.
It is important that the semen be protected from sunlight and that it be held at nearly constant animal body temperature from the time that it is collected until laboratory analysis is completed. All apparatus and containers that will come into contact with semen that is to remain viable must be clean and warmed to body temperature before use. A sample of a uniform mix of the collected gel-free semen is drawn and prepared for laboratory analysis. During analysis, the bulk of the semen should be stored in a stabilized incubator which has been preset to the proper temperature (38 degrees C. for equines). As soon as possible, however, the stored semen should be mixed with a pre-warmed life extending blend of chemicals and antibiotics (extender) in preparation for the insemination procedures which should immediately follow completion of the laboratory analysis. If the semen is to be shipped or used at a later time on-premises, the semen-extender mix must be cooled according to a prescribed program as a means of further increasing the sperm longevity. For some species, the semen may be cooled and maintained at refrigerated temperatures (5 degrees C.) for several days allowing safe shipping over long distances. Alternately, some species' semen can be frozen at cryogenic temperatures (-196 degrees C.) and stored indefinitely.
The laboratory work must be conducted with great care and be accomplished as rapidly and efficiently as possible. This is necessary to assure good correlation of the laboratory measurements and provide insemination fluid of the highest possible fertility.
The laboratory evaluation of the semen may be quite extensive. However, in routine work a minimal evaluation may be all that is required. A minimal evaluation includes at least the following six measurements and calculations:
1. An initial visual and olfactory examination to appraise semen quality and detect the presence of contaminants (dirt, urine, blood, etc.). PA1 2. A measurement of the total volume of ejaculate. PA1 3. An estimate of semen fertility. PA1 4. A determination of the spermatozoa concentration or density, i.e., the number of sperm cells per unit volume. PA1 5. A calculation of the total number of sperm cells per ejaculation. PA1 6. A determination of the volume of gel-free semen needed for each insemination. PA1 7. A calculation of the number of doses per collection. PA1 8. A recommendation for the volume of extender per dose and the total dose volume. PA1 9. A computation of the dilution ratio. PA1 10. A calculation of the total sperm per dose. PA1 11. A determination of motile sperm concentration. PA1 Ve is expressed in milliliters and C in millions of sperm cells per milliliter. PA1 Vd is the dose or volume of semen in milliliters (ml) to be used for insemination, PA1 N is the total number of viable spermatozoa desired for insemination in millions, PA1 C is the measured concentration or density of sperm in millions of cells per ml, and PA1 M is the estimated percentage of sperm that are viable (usually percent forward motility) expressed as a decimal fraction. PA1 Vc is the total volume of collected gel-free semen in milliliters and Vd is the volume of gel-free semen per dose in milliliters, computed using the equation of item 6. PA1 Vd is the volume of gel-free semen per dose in milliliters and Ve is the selected volume of extender per dose in milliliters. PA1 Ve is the volume of extender per dose in milliliters and Vd is the volume of gel-free semen per dose in milliliters. PA1 C is the measured sperm concentration in millions of cells per milliliter and Vd is the volume of gel-free semen per dose in milliliters. PA1 N is the total number of motile cells per dose in millions and Vt is the total volume of semen and extender per dose in milliliters.
The following additional evaluations are needed for a cooled and shipped and frozen semen programs and are often useful for on-premises breeding programs:
Items 1 and 2, sensory evaluation and measurement of volume, require no additional comment for purposes of this application.
Item 3, estimation of sperm fertility, is accomplished by a subjective microscopic examination of a standardized dilution of semen to determine the percentage of sperm that are viable. In routine practice, this is done by observing the motility patterns of the spermatozoa and estimating the percentage that move in a typical progressive or forward manner. Other types of movement that have been categorized are circular, vibratory, reverse and flagellating. These latter movement patterns are thought to be indicative of sperm cells that are unlikely to be capable of achieving ovum fertilization. A more detailed evaluation is sometimes used which includes a study of cell morphology under high power magnification. The extent to which the sample is free of cells having certain physical deformities which are thought to affect fertility may be used to refine the fertility rating.
Recently, with ongoing improvements in video and computer technology and the successful marriage of the two, instrumentation has been developed that will make statistically accurate objective measurements of sperm motility. At present, this equipment is used primarily at animal reproduction research facilities. Application at commercial breeding facilities is rare because of the high cost of the equipment compared with that of traditional instrumentation.
Item 4, determination of spermatozoa density or concentration C, may be accomplished by any of the several different methods, four of which are: (1) estimation based upon visual observation of semen turbidity under standardized conditions; (2) visual count using a microscope and hemocytometer; (3) electronic counting; and (4) photoelectric techniques in which a measured optical parameter of a standardized sample is correlated to sperm cell density.
Each method has particular advantages and disadvantages. Visual estimation is rapid and inexpensive but yields the poorest accuracy. Hemocytometer counts can be accurate but are tedious and time consuming. Properly performed electronic counting using modern instrumentation probably provides the highest accuracy but the equipment is expensive and the technique seems better suited to laboratory research and to the gathering of calibration data than to practical field use. Photoelectric instrumentation appears to offer the best compromise when factors of accuracy, rapidity of measurement, required operating expertise, cost and equipment portability are all considered and therefore has received a high degree of acceptance.
Item 5, total sperm per ejaculate Se, is simply the product of ejaculate volume Ve and sperm density C or: EQU Se=Ve.times.C million sperm cells
Where:
Total sperm per ejaculate may be used as a figure of merit for the male as a breeding animal because it represents his total sperm production capability, especially if it is averaged over a season.
Calculation of the volume of gel-free semen needed for each insemination, Vd, item 6, is done by substituting measured and selected values into a simple equation as follows: EQU Vd=N/(CM))
Where:
The total number of viable sperm cells desired for insemination, N, is the minimum number estimated to yield a high probability of achieving ovum fertilization. The selected quantity depends upon the species of animal, any knowledge of the probable degree of fertility of a particular female (from previous experience), and the expertise and judgement of the AI practitioner. As an example, the selected total number of viable sperm cells for insemination of a mare would normally fall within the range of 100 million to 500 million for on-farm breeding programs.
The sperm cell concentration or density, C, and the percent forward motility, M, are both taken from data gathered in the laboratory analysis of the collected semen.
Item 7, the total number of doses per collection Dc, is found from the relationship: EQU Dc=Vc/Vd doses
Where:
The recommended volume of extender per dose, VE, item 8, is based upon field experience and controlled experiments. For on-premises use, a quantity of 10 milliliters of extender is usually specified for equines unless the ratio of extender to semen would be less than 1:1. For cooled shipping the extender is increased to provide a total insemination dose of approximately 40 ml with a minimum dilution ratio of 3:1 (a higher ratio is used for shipped semen to increase sperm longevity). Experimentation with equines, indicates that total dose volumes in excess of 50 milliliters can result in decreased pregnancy rates. Similar considerations, with differing numerical factors, may apply to other animals. Obviously, depending upon sperm concentration and percentage of forward motility, there are situations that will not allow all of the above criteria to be met. In these cases, the judgement of the practitioner (ideally based upon previous experience with the particular animals being bred) is needed to determine the compromise that should be used to optimize the results.
The total volume of the insemination dose Vt, is given by: EQU Vt=Vd+Ve milliliters
Where:
The dilution ratio, Dr, verbally defined in the previous paragraph and listed as item 9, may be expressed mathematically as: EQU Dr=Ve/Vd
Where:
Item 10, represented by Sd, is the total number of spermatozoa per dose without regard to the progressively forward motility. This parameter is particularly useful when working with cooled or frozen shipped semen. Determination of the number of motile cells per dose of semen, as received after shipping, may be found by simply multiplying Sd by the measured percent forward motility of a semen sample taken after the recovery process (proper thawing or warming) has been completed. SD may be found from the relationship: EQU Sd=C.times.Vd million cells
Where:
Motile sperm concentration, Cm, Item 11, is the number of motile sperm per milliliter of dose, determined after the addition of extender. This parameter is also mainly of importance when working with cooled or frozen shipped semen. The motile sperm concentration may be found from the relationship: EQU Cm=N/Vt million cells per milliliter of dose
Where:
A review of the eleven steps required for a minimal evaluation of the collected semen shows that most are simple measurements or calculations or are subjective evaluations. Only two items require or lend themselves to the use of sophisticated instrumentation as a means of improving the rapidity and accuracy of semen evaluation. One of the items, estimation of semen fertility, can be enhanced through the use of a high quality phase-contrast type microscope. The evaluation, of course, remains subjective. Objective fertility ratings can be obtained from automated measurements of forward motility using computerized video equipment if the cost of the apparatus is not considered to be prohibitive. As alluded to earlier, the second item that can be instrumented is the measurement of spermatozoa density. A photoelectric method for estimating sperm concentration in semen was first reported in 1939 by Comstock and Green for the ram.
Several scientific studies have since been made by other investigators to determine the relationship between the sperm concentration of various domestic animals and such photometric parameters as light scattering (or haze), transmission and absorbance. Both duplicate hemacytometer counts and electronic counting have been used to establish the independent variable.
Before measurement, the semen is diluted by a standardized ratio. Various diluents have been used, most consisting of distilled water with a small percentage of sodium citrate or sodium chloride to balance osmotic pressures on the sperm cells. In addition, some investigators have advised the inclusion of formalin to kill and fix the spermatozoa. The standardized ratio of semen to diluent is initially determined by experiment. It is dependent upon the average semen turbidity of the species and the type of instrument to be used. The dilution ratio is chosen to optimize the spread of readings over the usable range of the selected instrument.
The investigations revealed that no photometric parameter could be considered a truly linear function of sperm concentration over the full gamut of densities that might be encountered. However, for several species absorbance was discovered to be tolerably linear over a rather wide practical range of median concentrations, with slope changes being evident usually toward both extremes of the scale. Acceptable accuracy could often be achieved by assuming a linear relationship for a practical range of values followed by smoothing the measurement deviations by using a linear least squares regression analysis to derive a best-fit calibration equation.
A technique that has been devised to provide improved accuracy where needed is to specify two or more dilution ratios with corresponding linear regression equations. In effect, this forms multiple overlapping ranges with a portion of each range falling over the most linear segment of the instrument characteristic. A subjective evaluation of semen turbidity is then used to select the dilution ratio-equation set that will optimize the measurement accuracy.
Another research finding was that the dominant radiation wavelength used for absorbance measurement is not a critical factor, at least over the range of the visible spectrum (380 nm to 760 nm). Absorbance simply decreases in a smooth and fairly gradual manner as wavelength is increased. This indicates that any wavelength of convenience (or even a wide band of wavelengths) may be used providing that measurements are made under the same conditions that were established for calibration of the instrument.
For a number of years, objective measurements of sperm concentration have been successfully accomplished in the field using specially calibrated general purpose laboratory spectrophotometers. Most of the instruments in use are analog types which were primarily designed for measurement of percent transmission of light through test samples at a selected (narrow band) wavelength. The more modern of these instruments include an absorbance scale which is logarithmically related to percent transmission (absorbance equals 2 minus log 10 of the percent transmission). Because a linear transmission scale can usually be interpolated and read more accurately than a non-linear absorbance scale, many of the operating procedures in use are based upon measurement of transmission. Conversion to absorbance is accomplished mathematically by the operator or, more conveniently, may be part of a computerized program if the sperm density value is taken from computer derived graphs or charts showing transmission as the independent variable.
In 1982 an investigation and analysis of commercially available photometers was conducted. None were found to be specialized (or even particularly well suited) for efficient use in the measurement of sperm density. This led to the development of a sperm Densimeter (U.S. Pat. Nos. 4,589,774 and 4,632,562) which has been manufactured for several years by the Animal Reproduction Systems Division of Dupree, Incorporated, assignee of the above referenced patents. This instrument is basically an analog design with digitization of some of the internal functions. It succeeded in providing improved efficiency and accuracy over prior methods of measurement and was well received by the industry. The core features and advantages of the original Densimeter include: A direct digital display of sperm density, thus eliminating the need for reference to charts or graphs; considerable improvement in accuracy through the use of direct absorbance measurement coupled with a three-segment data curve fit, which also eliminated the need for use of multiple dilution ratios; a broad spectrum low power light projection system for improved reliability; an insemination dose computer featuring digital display of the calculated dose, automatic entry of the measured sperm density, and digital thumbwheel entry of both percent forward motility and total viable sperm per dose. The stability of the original Densimeter is good and, although several steps of adjustment and a set of five calibration standards having known absorbance values are required for accurate calibration, the adjustments are independent, non-iterative and seldom required.
It is an object of the present invention to provide an instrument which functions on the basis of a transmittance measurement and which automatically performs zero and full scale transmission measurements for standardization.
It is an additional object of the present invention to provide a new and improved instrument which is microprocessor based and which provides improved reliability, superior accuracy, simplified operating procedure, greater versatility, elimination of manual calibration, and the availability of "expert advice" to aid the practitioner as needed in the decision making process.
It is an object of the invention to provide an instrument for measuring sperm concentration and calculating dose value and having an optical assembly with a light source, a specimen holder and a photodetector, a computation means, a display, and storage means for storing a concentration calculation equation, with the computation means including means for calculating sperm concentration using the stored concentration calibration equation and a sample transmittance value.
A further objective of the invention is to provide an optical assembly having means for measuring transmission of the specimen holder while empty with the light source energized, TFS, sample transmission of the specimen holder with a semen specimen with the light source energized, TS, and dark cell output of the photodetector with the light source energized, DC, with the computation means including means for calculating actual sample percent transmission as follows (TS-TDC)/(TFS-TDC).
An additional object of the invention is to provide an instrument wherein the means for measuring includes means for making a plurality of sample transmission value measurements in sequence and calculating an average for the plurality of sample transmission value measurements. A further object is to provide an instrument having means for providing the concentration calculation equation in the form of a multi-term polynomial derived from a plurality of data points relating measured sperm concentration and measured sperm transmittance value.
Another object of the invention is to provide an instrument wherein the computation means includes means for calculating actual volume of semen to be used as a dose, Vd, as follows Vd=N/(CN), where N is total number of viable spermatozoa desired, C is calculated sperm concentration, and M is estimated percentage of sperm that are viable. A further object is to provide an instrument wherein the light source includes a low power, lens-end light emitting diode producing a beam of narrow-band red light with a dominant wave length of 660 nM collimated with a collimating tube, and with an optically unfiltered silicon photodetector responsive to visible light in the red portion of the spectrum.
Further objects of the invention include calculation of number of insemination doses per collection, recommended volume of extender, total insemination volume, dilution ratio, total sperm dose, and motile sperm concentration and provision of additional calibrations for alternate animals (bulls, boars, rams, endangered species, etc.) and processing protocols (immediate use, cooled shipment, frozen, etc.) to be transferred into the densimeter by connecting the densimeter's serial port to a personal computer and running a loader program. Once loaded, the operator may select any of the new species calibration/processing protocols in addition to the original stallion semen processing protocols.
An additional object is to provide a densimeter with a built in "expert system" which can automatically optimize the number of motile sperm cells, number of insemination doses per collection, recommended volume of extender, total insemination volume, dilution ratio, total sperm per dose, and motile sperm concentration based on selected usage protocol.
Other objects, features and results will more fully appear in the course of the following description and claims.